We have constructed various baculovirus recombinants expressing selected rotavirus proteins including VP4 (P1A, P1B, P2, P3, P4, P5B, P6 or P7), VP7 (G1, G2, G3, G4, G6 or G9) or NSP4 (genotype A, B, C or D). Last year, by using immunocytochemistry assay involving such recombinants, we analyzed sera obtained from gnotobiotic calves or piglets infected orally with homologous bovine (NSP4[A]) or porcine (NSP4[B]) virus. We reported that following primary infection and subsequent challenge with virulent rotaviruses, naive calves and piglets developed either higher or significantly higher IgA and/or IgG antibody titers to the homologous-host NSP4[A] or [B] proteins than those to the heterologous-host rotavirus NSP4s, indicating that primary and secondary antibody responses were not NSP4 genotype-specific. Unexpectedly, such isotype antibody responses were shown to be correlated closely with the molecular phylogenetic relationships of NSP4 proteins within a species-specific region of amino acids 131-141, suggesting that NSP4-specific antibody responses were primarily species-specific. In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against oral challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, as a potential approach to immunization with a live oral rotavirus vaccine, the NSP4 protein did not appear to be important in protection against rotavirus disease and infection. This year, we constructed additional baculovirus recombinants expressing the VP4 (P8) and VP7 (G8) and by using these and other recombinants analyzed archival serum samples obtained from calves which were collected every month from birth to one year-old (12 samples/calf). Analysis of data is underway.